Although it has been known for decades that benzene is a human carcinogen, the mechanism by which benzene causes human diseases is still unknown. One reason for this lack of understanding is the complex metabolism of benzene, which gives rise to many electrophilic intermediates capable of binding with macromolecules [notably, 1, 2- and 1,4-benzoquinone (BQ), benzene oxide (BO), benzene diol (epoxide (BDE), 1,2- and 1,4-benzoquinone (BQ) and the muconaldehydes (MAHs)]. In addition, some metabolites generate reactive oxygen species (ROS) which can also cause toxic effects. We previously developed cysteinyl protein adducts as biomarkers of exposure to BO and the BQs and now propose to extend the work to include BDE and the MAHs. We will also apply existing methods to measure benzene and its metabolites in human urine and protein adducts associated with ROS in vivo (lysine adducts of malondialdehyde and 4-hydroxynonenal, the principal aldehyde product of lipid peroxidation). Armed with a comprehensive set of benzene biomarkers, we will explore the shapes of the gamut of exposure biomarker relationships in 1225 persons, about half of whom were occupationally exposed to benzene over a wide range of air levels (0.1 > 100 pp,). This will allow us to investigate the kinetics of the various metabolic routes. We will use these biomarkers to explore the magnitudes of inter-individual variability in human metabolism and to evaluate the association between biomarkers and genotypic expression of several enzymes involved in benzene metabolism (CYP4502E1, GSTT1 & GSTM1, NQO1, EH and MPO). We will also employ protein adducts to study the disposition of reactive benzene metabolites in rats and to compare the doses of these short-lived compounds in humans and rats, thereby improving quantitative risk assessment. Finally, we will use samples of blood and urine from unexposed persons to investigate the sources of background adducts of BO, the BQs and, potentially, to BDE and the MAHs. Collectively, these studies will provide information with which to characterize human metabolism of benzene, particularly in the critical range of benzene exposure below 10 ppm. Such information will be immensely valuable to those engaged in risk assessment and to researchers investigating the mechanism of benzene's particularly in the critical range of benzene exposure below 10 ppm. Such information will be immensely valuable to those engaged in risk assessment and to researchers investigating the mechanism of benzene's human health effects.